DETECTION OF THE IN-VIVO INCORPORATION OF A METAL CLUSTER INTO A PROTEIN - THE FEMO COFACTOR IS INSERTED INTO THE FEFE PROTEIN OF THE ALTERNATIVE NITROGENASE OF RHODOBACTER-CAPSULATUS

The photosynthetic bacterium Rhodobacter capsulatus has, in addition t o the Mo nitrogenase, a second Mo-independent nitrogen-fixing system, an 'iron-only' nitrogenase which is strongly repressed by molybdate. T he MoO42- concentration causing 50% repression of the alternative nitr ogenase in nifHDK- cells was 6 nM. If MoO42- was added to a growing ni fHDK- culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulat ed dramatically. In spite of the fact that C2H4 formation decreased co ntinuously during the duration of the experiment (3 days), the total C 2H6 Production increased about twofold within the first 24 h, whereas the relative yield of C2H6 increased from 2% (C2H6/C2H4 X 100) in the absence of MoO42-, to a maximal value of 69% in the presence of MoO42- (1 mM) after 72 h incubation. This 'Mo effect' appeared to be stronge r the higher the MoO42- concentration in the medium and the longer the incubation time. In the presence of ReO4-, WO42- or VO43-, a similar effect did not occur. The 'Mo effect' was not observed in a nifHDK-nif E- double mutant which is unable to synthesize the FeMo cofactor and w as diminished in a nifHDK-nifQ- mutant. Crude extracts from nifHDK- ce lls cultivated in the presence of MoO42-, also showed enhanced product ion of ethane. Component 1, purified from those extracts, displayed an S = 3/2 EPR signal which was relatively weak but characteristic for t he FeMoco. These results strongly support the suggestion that the 'Mo effect' is a consequence of the formation of a hybrid enzyme consistin g of the apoprotein of the alternative nitrogenase and the FeMo cofact or of the conventional nitrogenase. The 'Mo effect' was not influenced by the addition of chloramphenicol to the cultures. The occurrence of the 'Mo effect' appeared, therefore, to be independent of de-novo pro tein synthesis. The analysis of nifE-lacZ and nifN-lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also ex pressed under conditions of MoO42- deficiency. The possible explanatio ns for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.